Written by: Kathleen Cunningham
Written By: Ken Estrellas
Original Article: A. Mayeuf-Louchart et al. 2018
The Gist of It
Counting different colors, locating hundreds of dots, and measuring thousands of round shapes—these repetitive tasks are a large part of what scientists studying muscle have to do on a daily basis. Whether a scientist is trying to gain a better understanding of aging or look for the next treatment for childhood muscle diseases, the nuts and bolts of what they have to do are the same. These tasks include counting different types of proteins in muscle, locating muscle stem cells, and measuring the size of muscle fibers. Different types of muscle fibers are marked by any number of proteins and can be labeled with numerous colors. Muscle stem cells are relatively rare, but scientists need to be able to identify them among thousands of other cell types in tissue samples. Until recently, the size of muscle fibers, which indicate whether a muscle is growing or dying, had to be measured one at a time. The authors of a recently published paper describe a new computer program named MuscleJ that can perform these tasks automatically. After uploading a high-quality image of a cross-section of muscle, a research can use MuscleJ to automatically count different muscle markers in a sample, identify and count the number of muscle-specific stem cells, measure the sizes of all the muscle fibers in an image, and determine whether these muscles are degenerating (dying) or regenerating (growing). By automating and standardizing these repetitive tasks, MuscleJ can help save time and reduce human error in the field of muscle research. With this tool, scientists could potentially analyze samples faster and with greater consistency, helping speed up the time it takes to test drugs that could potentially be used to treat muscle diseases or traumatic muscle injuries.
The Nitty Gritty
In this study, Mayeuf-Louchart et al. report on the development of MuscleJ, an automated image analysis macro implemented in the open-source image analysis environment Fiji. The program is capable of identifying pre-specified regions of interest in muscle cross-section images and using them to automatically calculate and measure several, including muscle fiber morphology (in terms of area and diameter mean as well as fiber distribution), numbers of centrally vs. peripherally located myonuclei, percentages of peripherally nucleated (unaffected) vs. centrally nucleated (injured or regenerating) muscle fibers, numbers of satellite cells (peripherally located muscle progenitor cells marked by the cell surface marker Pax7), and fiber typing (up to three intrafiber stainings). Blood vessels can also be identified and measured in MuscleJ. In order to benchmark the program against manual analysis, five independent experts from two independent skeletal muscle biology labs evaluated a set of muscle cross-section images alongside MuscleJ, revealing a high level of agreement between both sets of results. Automated analysis with MuscleJ was 10-30 times faster than manual analysis depending upon the specific task, and multiple images can be analyzed with batch processing, allowing for standardized high-throughput analysis of large groups of skeletal muscle cross-sectional images.
Original Research Article: Alicia Mayeuf-Louchart, David Hardy, Quentin Thorel, Pascal Roux, Lorna Gueniot, David Briand, Aurélien Mazeraud, Adrien Bouglé, Spencer L. Shorte, Bart Staels, Fabrice Chrétien, Hélène Duez, and Anne Danckaert. “MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool.” Skeletal Muscle 8.1 (2018): 25.
Written By: Rebecca Tweedell
Original Article: Quemeneur et al. Infect Immun 2018
The Gist Of It
Time to talk about everyone’s favorite topic: diarrhea. I know, it’s gross and uncomfortable, but it’s also incredibly common. Have you ever gotten diarrhea while taking antibiotics? It’s a fairly frequent side effect that can be caused by a little bug known as Clostridium difficile, or C. diff for short. C. diff is a toxin-producing bacterium that is responsible for up to 25% of antibiotic-associated diarrhea and is also known to cause diarrhea in hospitalized patients. Since these infections are occurring in people who are already sick, they can be quite dangerous and even deadly. On top of that, antibiotic resistance is growing among C. diff, making the infections even harder to treat. The best way to treat a C. diff infection is to prevent it in the first place. That’s where vaccines come in. Recently, a vaccine that targets the toxins from C. diff was made. It has already been shown to be safe in people, but its ability to prevent infection is still unclear. The main concern is that the vaccine was made to target a specific C. diff bacterium. Just as we are all humans, but are each unique individuals, one C. diff bacterium can be quite different from another. So, in much the same way that different people react differently to the same medication, different C. diff individuals could be targeted differently by the same vaccine. A recent paper in the journal Infection and Immunity looked at how well the vaccine could work against 165 different C. diffs isolated from different infections. Fortunately, they found that this C. diff vaccine could neutralize all 165 different types. This is really exciting news because it means that this single vaccine could protect people from lots of potential C. diff infections, not just the one specific individual C. diff the vaccine was designed to target. This is an important step forward in the fight against C. diff infections, especially in the age of growing antibiotic resistance.
The Nitty Gritty
Quemeneur et al. began their study by characterizing different C. difficile toxinotypes. Using whole-genome sequencing, the researchers sequenced the toxin A and B genes from 49 of the 165 clinical isolates, representing 8 of the different toxinotypes (toxinotypes 0, I, III, IV, V, VI, VIII, and XII). As expected, they found high degrees of sequence conservation between strains within each toxinotype. They also found high degrees of conservation between toxinotypes 0, I, and XII. They went on to immunize hamsters with the C. difficile toxoid vaccine candidate, which is based on formalin-inactivated toxins A and B from the C. difficile reference strain VPI 10463. They tested the ability of the pooled sera from 12 hamsters to neutralize the cytotoxic activity of the 165 clinical isolates using cytotoxicity assays. These assays involved IMR-90 cells and the RCTA xCELLigence system, which uses gold microelectrodes in microtiter wells to monitor electric impedance, or the amount of the electric current flow that is impeded by the presence of cells (more cells = more electric impedance; more cell death from cytotoxicity = less impedance). In these assays, they found that the hamster sera could significantly neutralize cytotoxicity from all 165 clinical isolates. Furthermore, they found that this neutralization occurred regardless of the strain’s toxinotype or baseline level of cytotoxicity. The researchers went one step further and used a hamster challenge model to test the in vivo efficacy of the vaccine against the five most common toxinotypes. In this setting, the vaccine significantly protected hamsters infected with toxinotypes 0, III, IV, V, or VIII from symptoms and death.
Original Research Article: Quemeneur, Laurence, Nadine Petiot, Nadège Arnaud-Barbe, Catherine Hessler, Patricia J. Pietrobon, and Patricia Londoño-Hayes. “Clostridium difficile toxoid vaccine candidate confers broad protection against a range of prevalent circulating strains in a nonclinical setting.” Infection and Immunity 86.6 (2018): e00742-17.
Written by: Kaitlyn Sadtler
Original Article: J Godwin et al. 2013
The Gist of It
We all had a friend growing up, or maybe we were that friend, that had a lizard as a pet. In the USA in the 1990’s, when I was a kid, quite a few people had anoles. These little lizards were adorable, and something that blew my mind as an 8-year-old was, if they got stuck, and lost a piece of their tail, it would grow back – as if nothing happened. Some lizards even use their own tail as food in times of starvation. As a kid it was so cool that these little, humble, animals could lose a limb and grow it back, or “regenerate” it. Maybe that led me to studying regenerative medicine, which is the field of how we can try to help regenerate or re-grow missing or damaged tissues, like skin, muscle, or bone. In 2013, an amazing paper came out in the journal, Proceedings of the National Academies of the Sciences – PNAS for short. Here, they were looking at axolotls. Which are funny looking salamanders with their gills on the outside so they sort of look like they have a mane like a lion. Anyways, these salamanders can completely regenerate their arm. As with those little lizards who lost their tails, if an axolotl loses their arm, it grows back to normal in a few weeks. What was interesting in this study, was that if these axolotls didn’t have macrophages (a type of immune cell that we normally think of as fighting off infections) then, their arm doesn’t grow back, and instead you just have a stump with scar tissue. Here, they were able to show that this specific immune cell type was absolutely critical for axolotls to re-grow their arm. Now, why should we care about a salamander? The thing is, we can learn a lot from these animals that can help out both us humans, as well as other animals. If we know what makes animals regenerate that have these capabilities already (like salamanders), we could then turn them into therapeutics for humans.
The Nitty Gritty
In this study Godwin et al. we able to show that in the absence of macrophages, adult salamanders could not regenerate their forelimbs. To deplete macrophages, they injected clodronate liposomes into the limb bud. Clodronate is cytotoxic, therefore upon phagocytosis, they kill the cell that has ingested the liposomes carrying clodronate. If macrophages are depleted early in the regeneration process, a limb bud fails to develop and no mature tissue forms. Alternatively, if they are depleted later in regeneration, the process halts and an immature tissue structure remains. Histologically, we see dense extracellular matrix, characterized by large amounts of mature collagen I. In addition to increased collagen I in the limb bud of salamanders without macrophages, they also observed increased amounts of collagen IV and alpha smooth muscle actin, which is a marker of pro-fibrotic myofibroblasts. After this point, even if macrophage populations are allowed to return to a steady state, the limb fails to develop. However, if the scarred tissue is surgically removed, then the limb will complete the regenerative process.
Original Research Article: Godwin, James W., Alexander R. Pinto, and Nadia A. Rosenthal. “Macrophages are required for adult salamander limb regeneration.” Proceedings of the National Academy of Sciences 110.23 (2013): 9415-9420.